Effects Of A Vascular Targeted Peptide (GX1) On Tumor Formation And Angiogenesis And The Underlying Mechanisms

【Background】In 1971, Folkman laid the groundwork for research on therapies that target tumor vascularization by proposing that angiogenesis is necessary for tumor growth and invasion, and this system could be a legitimate target for anti-cancer agents. Subsequent studies showed that tumor vessels expressed a bunch of specific molecules which were hardly expressed in normal vessels. The discovery of such heterogeneity of tumor vasculature might lead to the investigation and application of these specific molecules in tumor vascular targeted therapy, so as to improve the efficiency of anti-tumor treatment, as well as to minimize the side effects.Since then, many studies have been done, and the application of phage display technique significantly facilitates the researches in this field. Currently, several candidate molecules, including RGD, NGR, DMXAA, AVE8062, etc. are in their phaseⅠ/Ⅱclinical trials, and promising results are observed in various kinds of tumors, such as breast cancer, ovarian cancer and prostate carcinoma. However, no such vascular targeted molecule had been identified in gastric cancer until GX1 was found in our lab.By using in vivo phage display technology, a cyclic 9-mer peptide CGNSNPKSC homing specifically to vasculature of human gastric adenocarcinoma was obtained. Immunohistochemical staining showed positive signal of GX1-displaying phage in the vascular endothelium of human gastric cancer, and GX1 expression level was negatively correlated with tumor differentiation, while no positive staining was observed in heart, liver, muscle, spleen or normal gastric tissues. In another study, GX1 was labeled with 99TcmO4- for ECT scanning to detect the in vivo distribution of 99Tcm-GX1. The imaging results showed that GX1 could concentrate in tumor xenograft in nude mice. All these results indicated that GX1 was a novel vascular target of human gastric cancer. However, there is still a lot of work to be done before GX1 become possible for clinical anti-tumor therapy as a tumor vascular targeted agent. The present study is to investigate the bioactivities and relevant molecular mechanisms of GX1 other than its targeting effect.【Objectives】1. To establish a co-culture model of tumor vascular endothelial cells for verification of the targeting effects of GX1 peptide (not the peptide displaying phage), and also as a platform for further tests.2. To study the in vitro effects of GX1 on proliferation, migration and other characteristics of vascular endothelial cells, and also to clarify the in vivo role of GX1 in tumor formation and angiogenesis.3. To investigate the possible mechanisms underlying the bioactivities of GX1, and to identify the specific signaling pathway and relevant molecules that may be regulated by GX1, so as to provide experimental evidence for clinical application of this targeted peptide.【Methods】1. HUVEC were co-cultured with tumor conditioned medium to establish the co-culture system of tumor endothelial cells (co-HUVEC). GX1 peptide, together with a control peptide (Pep2) was chemically synthesized and purified. Immunocytochemical staining was used to detect the binding specificity of GX1 to co-HUVEC.2. GX1 effects on cell proliferation, migration, vascular formation and other characteristics were analyzed by in vitro MTT assay, tube formation assay, wound healing test, etc. Chorioallantoic membrane (CAM) assay was used to observe GX1 effects on in vivo angiogenesis. Tumor formation test in nude mice bearing human gastric adenocarcinoma xenografts was performed with different drug administration modes of GX1.3. Cell cycle distribution and cell apoptosis induced by GX1 treatment were detected by flow cytometry to look into the possible mechanisms of GX1 effects on tumor angiogenesis.4. Microarray analysis was performed to detect the changes of gene expression induced by GX1 treatment in co-HUVEC. Bioinformatics analysis was used to identify the specific signaling pathway and downstream molecules which might be involved in GX1 functions.Western blot was used to determine the expression level of several genes significantly changed by GX1 treatment, including Bcl-2, BAX, caspase3 and caspase8, so as to verify the data from microarray analysis. 【Results】1. The co-culture system of tumor vascular endothelial cells (co-HUVEC) was successfully established, and GX1 and control peptide were synthesized and purified with the purity over 95%. GX1 was shown by immunocytochemical staining to bind specifically to co-HUVEC, while no positive staining was observed in Pep2 or PBS groups, indicating that GX1 could selectively target gastric cancer vasculature.2. MTT assay showed that compared with control peptide, GX1 significantly inhibited the cell proliferation of HUVEC and co-HUVEC in a dose-dependent manner, and the inhibition rate in co-HUVEC was higher than that of HUVEC (60% vs. 25%-45%, P<0.05). In tube formation assay, GX1 significantly hampered the micro-tube formation of co-HUVEC, while no such effect was seen in Pep2 treated cells. On the other hand, GX1 showed no significant effects on cell proliferation or migration ability in tumor cells.3. CAM assay exhibited that, compared with Pep2, GX1 remarkably suppressed the neovascularization on CAM in chicken embryo (micro-vessel count: 20.0±1.6 vs. 10.6±1.0, P<0.05), suggesting that GX1 could inhibit in vivo angiogenesis. In in vivo tumor formation assay, GX1, either administered intravenously or subcutaneously, partly delayed tumor growth in comparison with Pep2 or PBS.4. Flow cytometry analysis showed that compared with Pep2, GX1 increased the proportion of apoptotic cells in co-HUVEC (1.8% vs. 10.5%, P<0.05), while no significant changes in cell cycle distribution was detected in GX1 treated co-HUVEC.5. Microarray data exhibited that, among the total of 39200 tested genes, 248 genes were notably upregulated, while 137 genes were downregulated by GX1 treatment. These genes were classified as genes associated with cell apoptosis, cell metabolism, oncogenes and tumor suppressor genes, etc. Western blot results confirmed the upregulated protein level of BAX and caspase3, but a downregulation of Bcl-2 after GX1 treatment of co-HUVEC. There was no significant change of caspase8 activity.【Conclusions】1. Tumor microenvironment can be effectively mimicked by in vitro co-culture system. HUVEC co-cultured with tumor conditioned medium exhibit some of the characteristics of tumor endothelial cells, and could be used in research of tumor vascular targeted molecules.2. Besides the tumor vascular targeting ability, GX1 is also able to inhibit tumor angiogenesis in vitro and in vivo, as well as partly suppress tumor growth in vivo possibly by induction of cell apoptosis of tumor endothelial cells.3. GX1 might induce cell apoptosis via regulation of Bcl-2/BAX signaling pathway and activation of caspase family members. These results may facilitate the development of GX1 as a novel candidate for vascular targeted therapy of human gastric cancer, and may also provide valuable information for identification of GX1 receptors.